We have made a preliminary observation that caspase-3 present in mitochondria from Jurkat cells, and that within ten minutes that Fas ligation, it is processed to a p29 form lacking the pro-domain. Over- expression of Bcl-2 prevents the appearance of caspase-3 in mitochondria and blocks Fas-mediated apoptosis in stably transfected CEM cells. Taken together, these findings suggest that caspase activation may occur within mitochondrial inter-membrane space, rather than in the cytosol. Furthermore, it suggests that signals from plasma membrane Fas are transmitted rapidly to mitochondria to stimulate caspase processing. By preparing mitochondria with fully intact outer membranes and through the use of differential sedimentation methods to isolate highly purified fractions of outer mitochondrial membranes, inter-membrane space components, and inner membrane/matrix components, as well as cytosol, we can test the hypothesis that pro-caspase-3 is processed initially in the mitochondria, and we can examine the role of caspase-8 and -9, Apaf-1, Bcl-2, and Bax in this process. We propose the following specific aims: 1) evaluate caspase-3 import into mitochondria using in vitro transcribed/translated caspase-3 and intact mitochondria from control and Fas-ligated cells; 2) Examine intramitochondrial processing of caspase-3. Delineation of these events is essential to understanding the fundamental mechanisms of apoptosis, which may lead to identification of new therapeutic targets for regulating apoptosis. The heart is an organ that is extremely rich in mitochondria and dependent upon their function; apoptosis has been shown to occur in heart failure as well as in ischemia/reperfusion and other myocardial disease processes; therefore our third specific aim will be to: 3) Evaluate cardiomyocytes for parallel events induced by simulated ischemia and reperfusion. Understanding these fundamental processes in the comparatively simple and easily manipulated system of Jurkat cells and Fas-mediated apoptosis can be readily translated to studies in the heart.